Forrest J Craig, Paden Clinton R, Allen Robert D, Collins Julie, Speck Samuel H
Department of Microbiology and immunology, Emory Vaccine Center, Emory University School of Medicine, 1462 Clifton Rd., Atlanta, GA 30322, USA.
J Virol. 2007 Nov;81(21):11957-71. doi: 10.1128/JVI.00111-07. Epub 2007 Aug 15.
Gammaherpesviruses establish lifelong, latent infections in host lymphocytes, during which a limited subset of viral gene products facilitates maintenance of the viral episome. Among the gamma-2-herpesvirus (rhadinovirus) subfamily, this includes expression of the conserved ORF73-encoded LANA proteins. We previously demonstrated by loss-of-function mutagenesis that the murine gammaherpesvirus 68 (MHV68) ORF73 gene product, mLANA, is required for the establishment of latency following intranasal inoculation of mice (N. J. Moorman, D. O. Willer, and S. H. Speck, J. Virol. 77:10295-10303, 2003). mLANA-deficient viruses also exhibited a defect in acute virus replication in the lungs of infected mice. The latter observation led us to examine the role of mLANA in productive viral replication. We assessed the capacity of mLANA-deficient virus (73.Stop) to replicate in cell culture at low multiplicities of infection (MOIs) and found that 73.Stop growth was impaired in murine fibroblasts but not in Vero cells. A recombinant virus expressing an mLANA-green fluorescent protein (GFP) fusion revealed that mLANA is expressed throughout the virus replication cycle. In addition, 73.Stop infection of murine fibroblasts at high MOIs was substantially more cytotoxic than infection with a genetically repaired marker rescue virus (73.MR), a phenotype that correlated with enhanced kinetics of viral gene expression and increased activation of p53. Notably, augmented cell death, viral gene expression, and p53 induction were independent of viral DNA replication. Expression of a mLANA-GFP fusion protein in fibroblasts correlated with both reduced p53 stabilization and reduced cell death following treatment with p53-inducing agonists. In agreement, accentuated cell death associated with 73.Stop infection was reduced in p53-deficient murine embryonic fibroblasts. Additionally, replication of 73.Stop in p53-deficient cells was restored to levels comparable to those of 73.MR. More remarkably, the absence of p53 led to an overall delay in replication for both 73.Stop and 73.MR viruses, which correlated with delayed viral gene expression, indicating a role for p53 in MHV68 replication. Consistent with these findings, the expression of replication-promoting viral genes was positively influenced by p53 overexpression or treatment with the p53 agonist etoposide. Overall, these data demonstrate the importance of mLANA in MHV68 replication and suggest that LANA proteins limit the induction of cellular stress responses to regulate the viral gene expression cascade and limit host cell injury.
γ-疱疹病毒可在宿主淋巴细胞中建立终身潜伏感染，在此期间，有限的一部分病毒基因产物有助于维持病毒附加体。在γ-2-疱疹病毒（嗜淋巴细胞病毒）亚科中，这包括保守的ORF73编码的LANA蛋白的表达。我们之前通过功能丧失诱变证明，小鼠γ-疱疹病毒68（MHV68）的ORF73基因产物mLANA是小鼠经鼻接种后建立潜伏感染所必需的（N. J. Moorman、D. O. Willer和S. H. Speck，《病毒学杂志》77:10295 - 10303，2003年）。缺乏mLANA的病毒在感染小鼠的肺部急性病毒复制中也表现出缺陷。后一观察结果促使我们研究mLANA在病毒增殖性复制中的作用。我们评估了缺乏mLANA的病毒（73.Stop）在低感染复数（MOI）下在细胞培养中的复制能力，发现73.Stop在小鼠成纤维细胞中的生长受损，但在Vero细胞中不受影响。一种表达mLANA - 绿色荧光蛋白（GFP）融合体的重组病毒显示，mLANA在整个病毒复制周期中都有表达。此外，高MOI下73.Stop感染小鼠成纤维细胞的细胞毒性比用基因修复的标记拯救病毒（73.MR）感染大得多，这一表型与病毒基因表达动力学增强和p53激活增加相关。值得注意的是，细胞死亡增加、病毒基因表达和p53诱导与病毒DNA复制无关。成纤维细胞中mLANA - GFP融合蛋白的表达与p53诱导激动剂处理后p53稳定性降低和细胞死亡减少相关。与此一致的是，在p53缺陷的小鼠胚胎成纤维细胞中，与73.Stop感染相关的加剧的细胞死亡减少。此外，73.Stop在p53缺陷细胞中的复制恢复到与73.MR相当的水平。更值得注意的是，p53的缺失导致73.Stop和73.MR病毒的复制总体延迟，这与病毒基因表达延迟相关，表明p53在MHV68复制中起作用。与这些发现一致，复制促进病毒基因的表达受到p53过表达或用p53激动剂依托泊苷处理的正向影响。总体而言，这些数据证明了mLANA在MHV68复制中的重要性，并表明LANA蛋白限制细胞应激反应的诱导，以调节病毒基因表达级联并限制宿主细胞损伤。
